RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to guide the inhibition of gene expression in a sequence-specific manner. Its principal role is suppression of potentially harmful genetic material (Buchon et al., “RNAi: a defensive RNA-silencing against viruses and transposable elements”, Heredity, 96(2), 95-202, 2006). Among the most powerful stimuli capable of triggering the RNAi machinery are long double-stranded ribonucleic acids (dsRNA) often associated with viral replication. These long duplexes are degraded into short double-stranded fragments (approximately 21-23 nucleotides long) known as small interfering RNA (siRNA) by an RNAse III-type enzyme, Dicer (Bernstein et al., “Role for a bidentate ribonuclease in the initiation step of RNA interference” Nature, 409, 363-366, 2001). The siRNA is then transferred onto the RNA-induced silencing complex (RISC) (Tuschl et al., “RISC is a 5′-phosphomonoester-producing RNA endonuclease”, Genes & Development, 18: 975-980, 2004) which becomes activated upon removal of one of the strands (the “passenger” or “sense” strand). The remaining strand (the “guide” or “antisense” strand) then directs the activated RISC in a sequence-specific degradation of complementary target messenger RNA (mRNA). Since the selection of engaged mRNA is controlled solely by Crick-Watson base-pairing (Watson, J. D, Crick, F. H “Molecular structure of nucleic acids”, Nature, (171), 737-738, 1953) between the guide strand and the target mRNA, the RNAi pathway can be directed to destruct any mRNA of a known sequence. In turn, this allows for suppression, or knock-down, of any gene from which it was generated preventing the synthesis of the target protein. This unprecedented control has wide reaching therapeutic consequences.
While long dsRNA will also inevitably trigger a sequence independent immunogenic reaction, much smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P.; “RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.” Cell, 101, 25-33, 2000). According to this, artificially synthesized 21 nucleotide long RNA duplexes typically containing 2-nt 3′-overhangs, can be used to manipulate any therapeutically relevant biochemical system, including ones which are not accessible through traditional small molecule control.
In order to realize this immense therapeutic potential of RNAi, many properties of siRNAs need to be optimized (Castanotto et al. “The promises and pitfalls of RNA interference-based therapeutics”, Nature, 457, 426-457, 2009). Due to their large molecular weight and polyanionic nature unmodified siRNAs do not freely cross the cell membranes, and thus development of special delivery systems is required (White, P. J. “Barriers to successful delivery of short interfering RNA after systemic administration” Clin. Exp. Pharmacol. Physiol. 35, 1371-1376, 2008). Equally important is optimization of potency (Koller, E. et al. “Competition for RISC binding predicts in vitro potency of siRNA” Nucl. Acids Res. 34, 4467-4476, 2006), stability (Damha et al. “Chemically modified siRNA: Tools and applications” Drug Discovery Today, 13(19/20), 842-855, 2008), and immunogenicity (Sioud “Innate sensing of self and non-self RNAs by Toll-like receptors”, TRENDS in Molecular Medicine, 12(4), 167-176, 2006).
The guide-strand-mediated sequence-specific cleavage activity of the RNA-induced silencing complex (RISC) is associated with an RNase H-type endonuclease Argonaute (Ago) (Tanaka Hall “Structure and Function of Argonaute Proteins”, Structure, 13, 1403-1408, 2005). An X-ray crystal structure of Argonaute 2 (Ago2) containing a chemically modified guide strand (Patel at al., “Structure of the guide-strand-containing argonaute silencing complex”, Nature, 456(13), 209-213, 2008, Patel at al., “Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex”, Nature, 456(13), 921-927, 2008) revealed that nucleotides 2 through 8 of the guide strand (referred to as “seed region”) are preassembled in a A-form helix and that the guide strand makes contact with the surface of the Ago2 through its sugar/phosphodiester backbone. This observation bodes well for the importance of the seed region in the initial recognition of the complementary mRNA, since an effective mRNA/guide strand interaction requires the heterobases to be accessible from the cytoplasm, hence to point away from the receptor surface.
Chemical modifications of the sugar/phosphodiester backbone of the siRNA's guide strand are therefore expected to have profound effect on the siRNA/Ago2 interaction. This offers a way to optimize the performance of this complex. Such an improved interaction should result in increased siRNA/Ago2 binding selectivity, more effective strand selection and passenger strand cleavage, improved catalytic turnover, siRNA/Ago2 complex stability and product release. Moreover, chemically modified siRNA duplexes are expected to be quite resistant to RNase mediated cleavage (increased half-life), decreased affinity to Toll-like receptors (TLR) and dsRNA-dependent protein kinase (PKR), resulting in decreased immunogenicity (Liang et al. “RNA Interference with Chemically Modified siRNA”, Cur. Topics Med. Chem., 6, 893-900, 2006).
Most chemically modified nucleosides used today in RNAi were synthesized to convey enzymatic stability to RNA oligomers and their design was not guided by siRNA/Ago2 binding considerations. Even though collection of SAR data relevant to this interaction would be highly beneficial, a hypothesis driven design of such novel nucleosides is clearly hampered by the chemical complexity of the associated chemistry. Assuming that the interaction of the siRNA's guide strand with the Ago2 surface is primarily static, such an effort would require independent SAR data collection for each nucleotide along the siRNA oligomer separately, since the local Ago2 surface relevant to each particular position is different. Furthermore, such SAR study would require the synthesis of sugar-modified nucleosides containing all four canonical bases since a systematic investigation would require a use of a sequence-specific siRNA. In theory, at least 21 separate siRNA oligomers, containing one instance the modified nucleoside each (positions 1 through 21, “walkthrough”) would be necessary, requiring the synthesis of considerable quantities of each monomer. This complexity renders the interrogation of such a huge chemical space intractable.
We have realized that use of universal bases in place of canonical heterocycles would greatly simplify the problem. In general, a universal base is a heterocycle capable of an isoenergetic interaction with each one of the canonical heterobases, (Adenine, Guanine, Uracil and Cytosine) while part of a double helix. The simplest example of such a universal interaction is a hydrogen atom corresponding to the removal of the heterocycle and more complex examples are 3-nitro-pyrrole, imidazole-4-carboxamide, 5-nitro-indole, inosine and others. We have argued that the use of such universal base would not only eliminate the need for synthesis of all four canonical nucleosides, it would also greatly reduce the complexity of the associated chemical syntheses.
Replacement of a canonical heterocycle with a universal base was expected to affect the efficiency of such a siRNA/Ago2 assembly and this should result in a position specific decrease of target gene knockdown. In order to asses this effect, we have synthesized ApoB-specific siRNA oligomers containing such universal bases while keeping the sugar of the nucleoside unmodified. We have indeed observed a base dependent position specific change in overall knockdown performance. In general, the effect of the universal base was more pronounced in the center of the RNA oligomer and the effect of simple base-surrogates such as hydrogen (removal of the entire heterocycle) was more profound.
Using the position-specific knockdown data obtained with universal base-surrogates as the new baseline, we synthesized a series of sugar-modified base-surrogate containing nucleosides and evaluated them under conditions similar to those used to obtain the baseline data. We argued that if the performance of the base-surrogate-containing sugar-modified nucleoside surpasses that of the base-surrogate containing canonical sugar, this relationship will be reflected also in the improved performance of the sugar-modified full canonical nucleoside and vice versa. This approach would allow us to rapidly evaluate prospective sugar modification and perform the syntheses of the full nucleosides only when the particular modification is found beneficial in this simplified platform. While this novel platform does not allow for a prediction of useful modifications, it can dramatically increase the rate of SAR data collection.